Polymerase chain reaction (PCR) amplification of a portion of the genome of both rapidly growing mycobacteria and nocardiae, followed by restriction fragment length polymorphism (RFLP) analysis of the amplification products, has proven to be a useful technique in the diagnostic laboratory. Identification of these organisms to the species level can be obtained within a few days of organism isolation, as compared with the month or more required for conventional identification based on biochemical testing. In addition, these molecular procedures allow more accurate discrimination among species and subspecies than is possible with biochemical testing. Our work with two different areas of the Nocardia genome (a portion of the gene for 16S ribosomal RNA and a portion of the gene for the heat-shock protein) has suggested the existence of hitherto unrecognized Nocardia species; work is ongoing to characterize these organisms further. In addition, we have found several clinical isolates belonging to species of Nocardia that have not, so far as we know, been previously reported to cause disease in patients in the western hemisphere. A manuscript describing the RFLP component of our methodology has been published. DNA-DNA hybridization is currently being utilized to define more precisely which of our isolates belong to unusual but already-described species, and which isolates might belong to hitherto undescribed species. A characterization of these isolates based on phenotypic and other molecular biologic features is also underway. The fact that some unusual Nocardia species can be pathogenic in patients with chronic granulomatous disease has also been noted in another manuscript.